Interaction of draxin and γ-netrins

ABSTRACT

This invention relates to extracellular protein-protein interactions and their possible therapeutic uses. More particularly, this invention describes the interaction between Draxin, particularly fragments binding to γ-Netrins comprising SEQ ID NO.: 1, 2 or 3, and variants thereof, with γ-Netrins, and the use of this interaction to disrupt γ-Netrin/Netrin receptor interactions. The invention also relates to diagnostic and/or therapeutic uses of Draxin or fragments or variants thereof, as well as to an antibody against Draxin inhibiting binding of Draxin to γ-Netrins. Further, the invention relates to fragments of γ-Netrins, in particular Draxin-binding Netrin1-fragments comprising SEQ ID NO.: 51 and variants thereof, as well as to an antibody against γ-Netrins inhibiting binding of γ-Netrins to Netrin receptors.

CROSS REFERENCE TO RELATED APPLICATION

This application is a 35 U.S.C. 371 National Phase Entry Applicationfrom PCT/EP2015/051088, filed Jan. 21, 2015, which claims the benefit ofU.S. Patent Application No. 62/049,643 filed on Sep. 12, 2014 andEuropean Patent Application No. 14152341.5 filed Jan. 23, 2014, thedisclosure of which are incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to extracellular protein-protein interactions andtheir possible therapeutic uses. More particularly, this inventiondescribes the interaction between Draxin, particularly fragments bindingto γ-Netrins comprising SEQ ID NO.:1, 2 or 3, and variants thereof, withγ-Netrins, and the use of this interaction to disrupt γ-Netrin/Netrinreceptor interactions. The invention also relates to diagnostic and/ortherapeutic uses of Draxin or fragments or variants thereof, as well asto an antibody against Draxin inhibiting binding of Draxin to γ-Netrins.Further, the invention relates to fragments of γ-Netrins, in particularDraxin-binding Netrin1-fragments comprising SEQ ID NO.: 51 and variantsthereof, as well as to an antibody against γ-Netrins inhibiting bindingof γ-Netrins to Netrin receptors.

BACKGROUND OF THE INVENTION

The laminin-related netrin protein family in humans comprises 5 members.Three of them, Netrin1, Netrin3, and Netrin4 are secreted proteins.NetrinG1 and NetrinG2 instead are linked to the cell surface by a GPIanchor. The LamNT domain and the EGF domains of Netrin1 and Netrin3 arederived from the γ chain of Laminin1. In the context of the presentinvention, these Netrins are thus referred to as “γ-Netrins”. Incontrast, the corresponding domains of Netrin4, NetrinG1 and NetrinG2are homologous to the domains present in the β chain of Laminin1 (Mooreet al., 2007).

Netrin1 is a diffusible, laminin-related protein identified as neuronalguidance cue during development of the nervous system. Netrin1 mediatesits biological effects through binding to receptors, which belong to theso-called dependence receptors, e.g. deleted in colorectal cancer (DCC)and uncoordinated-5-homolog (UNC5H). Recently, it has been found thatNetrin1 is expressed outside the nervous system and contributes to thepatterning of developing epithelial tissues such as mammary gland,pancreas, and lung by regulating diverse processes including adhesion,motility, proliferation, and differentiation of cells.

Numerous tumors have been described to express cell surface receptorsbelonging to the DCC-und UNC5-family. These receptors are binding to thesecreted ligand Netrin1 in the extracellular space and serve, inaddition to their well-established neurodevelopmental function, asdependence receptors in cancers (Castets et al., 2012; Mehlen et al.,2011). In tumors they can regulate tumor cell survival in a Netrin1dependent manner. Netrin1 itself is known to be upregulated by manytumor types and has been suggested to act as an oncogene (Arakawa, 2004;Fitamant et al., 2008). If Netrin1 is not bound to dependence receptorsof the DCC- and UNC5-family, the receptors cannot form dimers ormultimers, which in turn triggers the activation of a pro-apoptoticpathway.

In several studies, decoy Netrin receptor fragments have been used todisrupt Netrin/Netrin receptor interactions in order to inducepro-apoptotic signaling. For example, such receptor fragments have beenused in cancer cell lines (Delloye-Bourgeois et al., 2009; Fitamant etal., 2008) and in animal models (Fitamant et al., 2008; Paradisi et al.,2013; Paradisi et al., 2009) to induce cancer cell death. However, usinga fragment of a Netrin receptor causes interference at a relatively latestage of the signaling cascade, namely just before dimerization of thereceptor. Moreover, even when using high concentrations of these decoyreceptors, a residual binding of Netrin to the full length receptorcannot be prevented.

It was thus an object of the invention to provide compounds that can beused for interfering with the binding of γ-Netrins, in particularNetrin1 to at least one of its receptors, which at least partiallyovercome the disadvantages of the prior art.

Draxin is a secreted protein described to be involved in axon guidancedecisions (Islam et al., 2009). In contrast to Netrins 1-3, which arepresent in vertebrates and invertebrates, Draxin can only be found invertebrate genomes. The amino acid sequence of human Draxin is shown asSEQ ID NO.: 4. In zebrafish, there exist two Draxin isoforms (DraxinAand DraxinB); their amino acid sequences are represented by SEQ ID NO.:5 and SEQ ID NO.: 6.

By using an extracellular protein-protein interaction screen assay(AVEXIS), the present inventors identified Draxin as a novel directbinding partner for Netrin1. Furthermore, by using an AVEXIS basedcompetition assay, the inventors were able to show that Draxin or Draxinprotein fragments can compete with Netrin receptors for binding toNetrin1.

The present invention therefore provides specific peptides binding toγ-Netrins (“γ-Netrin-binding peptides”), particularly to Netrin1, aswell as antibodies directed against γ-Netrins (“γ-Netrin-bindingantibodies”), particularly against Netrin1, which may be used forinterfering with γ-Netrin/Netrin receptor binding, in particularNetrin1/Netrin receptor binding. The invention further providesDraxin-binding peptides as well as Draxin-binding antibodies inhibitingbinding of Draxin to γ-Netrins, in particular to Netrin1.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention relates to peptides which bind toat least one γ-Netrin, with a high specificity and a high affinity.Importantly, the affinity of Draxin to the γ-Netrins, in particular toNetrin1, is significantly higher than the affinity of γ-Netrins, inparticular of Netrin1 to Netrin receptors.

Accordingly, the invention provides a γ-Netrin-binding peptide,comprising (i) the sequence EVMPTLDMALFDWTDYEDLKP (SEQ ID NO.: 1), or(ii) the sequence DVAPTFNMALFDWTDYEDMRP (SEQ ID NO.: 2), or (iii) thesequence EVMPTLDMTLFDWTDYEDMKP (SEQ ID NO.: 3), or (iv) a variantthereof having a sequence identity of at least 70%, at least 80%, atleast 85%, at least 90%, or at least 95% to SEQ ID NO.: 1, 2 and/or 3,wherein said peptide has a length of up to 328 amino acids and isoptionally fused to a heterologous peptide or polypeptide. Preferably,the γ-Netrin is Netrin1.

The γ-Netrin-binding peptide is characterized in that it binds with highaffinity to Netrins derived from the γ chain of Laminin1, comprising inparticular human Netrin1 and human Netrin3 as well as Netrins 1a, 1b and2 from zebrafish (Danio rerio). In particular, the γ-Netrin-bindingpeptide of the invention binds to human Netrin1 and to zebrafishNetrin1a (also referred to as Ntn1a) and Netrin1 b (also referred to asNtn1b). In preferred embodiments, it is therefore referred to as“Netrin1-binding peptide”.

In some embodiments, the peptide with a length of up to 328 amino acidscomprises any one of SEQ ID NO.: 1, 2, 3 or a variant thereof having asequence identity of at least 70%, at least 80%, at least 85%, at least90%, or at least 95% to SEQ ID NO.: 1, 2 and/or 3 and is free of anyfurther heterologous peptides.

In other embodiments, the peptide with a length of up to 328 amino acidscomprises any one of SEQ ID NO.: 1, 2, 3 or a variant thereof having asequence identity of at least 70%, at least 80%, at least 85%, at least90%, or at least 95% to SEQ ID NO.: 1, 2 and/or 3 and is fused to aheterologous peptide or polypeptide as defined below.

The minimal sequence of the γ-Netrin-binding peptide, in particularNetrin1-binding peptide is represented by

SEQ ID NO: 1 EVMPTLDMALFDWTDYEDLKP, SEQ ID NO.: 2 DVAPTFNMALFDWTDYEDMRP,and SEQ ID NO.: 3 EVMPTLDMTLFDWTDYEDMKP, respectively.

In some embodiments, the peptide has a length of up to 328 amino acids,up to 324 amino acids, up to 320 amino acids, up to 250 amino acids, upto 200 amino acids, up to 100 amino acids, up to 50 amino acids, or upto 30 amino acids. According to other embodiments, the peptide has alength of up to 150 amino acids or up to 125 amino acids. Fragmentscomprising up to 50 amino acids are preferred according to someembodiments.

In certain preferred embodiments, a variant of SEQ ID NO.: 1, SEQ IDNO.: 2 or SEQ ID NO.: 3 have a sequence identity of at least 70% to SEQID NO.: 1, 2 and/or 3. In further embodiments the level of sequenceidentity may be at least 90%, or even at least 95% to SEQ ID NO.: 1, 2and/or 3.

A “variant” in the context of the present invention is any peptide whoseamino acid sequence varies in at least one position from the respectivereference peptide, but retains the biological activity of the referencepeptide; for example, a variant of SEQ ID NO.: 1 differs in at least oneamino acid therefrom, and retains the γ-Netrin-, particularlyNetrin1-binding activity. In particular, variants of SEQ ID NO.: 1, 2and 3 differ in 1, 2, 3, 4, 5, 6 or 7 amino acids from SEQ ID NO.: 1,SEQ ID NO.: 2 and/or SEQ ID NO.: 3, provided they retain the γ-Netrin-,particularly Netrin1-binding activity. Variations will usually begenerated by amino acid substitutions. Particularly, a variant accordingto the invention will be characterized in that it has been changed tocontain at least one non-naturally occurring substitution modificationrelative to the respective reference peptide.

The peptidic compounds of the present invention comprise a linearbackbone of amino carboxylic acids linked by peptide, i.e. carboxamidebonds. Preferably, the amino carboxylic acids are α-amino carboxylicacids and more preferably L-α-amino carboxylic acids, unless indicatedotherwise. Any amino acid of the sequences disclosed herein may bereplaced either by an unmodified canonical proteinogenic L-amino acid,or by an unmodified canonical proteinogenic D-amino acid. Also envisagedare substitutions with non-canonical proteinogenic amino acids, inparticular with ornithine, 2,4-diamino butyric acid, 2,3-diaminopropionic acid, selenocysteine, pyrrolysine, hydroxyproline,O-phosphoserine, O-phosphotyrosin, γ-carboxyglutamic acid,γ-aminobutyric acid, norleucine, ε-aminohexanoic acid, and with otherposttranslationally modified amino acids, e.g. amino acids with amidatedcarboxyl groups (at C-termini of peptides), amino acids with alkylated(e.g. methylated) side chains, amino acids with an amino side chaingroup (such as lysine and ornithine) with modifications at one or bothof the hydrogen atoms of the amino side chain group, for example with alipophilic moiety attached via a carboxamide bond, etc.

The percent sequence identity may be determined according to thefollowing formula:I=n:Lwherein I is the identity in percent, n is the number of identical aminoacids between a given sequence and a comparative sequence as shown e.g.in SEQ ID NOs.: 1, 2 and 3, and L is the length of the comparativesequence. Importantly, when calculating the percent sequence identityaccording to this formula, an alignment of the two sequences shall becarried out without gaps between complementary portions and over thewhole length of the comparative sequence.

In specific embodiments, the invention provides a γ-Netrin-bindingpeptide, comprising (i) the sequence EVMPTLDMALFDWTDYEDLKP (SEQ ID NO.:1), or (ii) the sequence DVAPTFNMALFDWTDYEDMRP (SEQ ID NO.: 2), or (iii)the sequence EVMPTLDMTLFDWTDYEDMKP (SEQ ID NO.: 3), or (iv) a variantthereof having a sequence identity of at least 70%, at least 80%, atleast 85%, at least 90%, or at least 95% to SEQ ID NO.: 1, 2 and/or 3,wherein said peptide has a length of up to 100 amino acids and isoptionally fused to a heterologous peptide or polypeptide.

According to some embodiments, the γ-Netrin-binding peptide, inparticular the Netrin1-binding peptide, comprises any one of SEQ ID NO.:7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ IDNO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14 or SEQ ID NO.: 15 or acorresponding fragment of another species. Preferably, the peptidecomprises any one of SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15 or a correspondingfragment of another species, more preferably SEQ ID NO.: 15 or acorresponding fragment of another species.

In some preferred embodiments, the γ-Netrin-binding peptide, inparticular the Netrin1-binding peptide, comprises a sequence which has alength of 22 amino acids. This 22-amino-acid (22aa) sequence may forexample be SEQ ID NO.:16, or it may be SEQ ID NO.: 14, or it may be SEQID NO.: 17, or it may be a variant of any of these sequences, e.g. acorresponding fragment from another species with an additional aminoacid residue, preferably glycine (Gly) at the N-terminus. Variants ofSEQ ID NO.: 16, 14, 17 have a sequence identity of at least 70% to SEQID NO.: 16, 14 and/or 17. In further embodiments, the level of sequenceidentity may be at least 90%, or even at least 95% to SEQ ID NO.: 16, 14and/or 17. In particular, a variant of SEQ ID NO.: 16, 14, 17 may differin 1, 2, 3, 4, 5, 6 or 7 amino acids from SEQ ID NO.: 16, SEQ ID NO.: 14and/or SEQ ID NO.: 17, provided they retain the γ-Netrin-bindingactivity.

Also encompassed by the invention is any variant of any one of SEQ IDNOs.: 7-15 or a corresponding fragment of another species having asequence identity of at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, or at least 95% thereto, as long as thebinding activity to γ-Netrins, preferably to Netrin1, is maintained. Insome preferred embodiments, the level of sequence identity is at least70%. In further preferred embodiments, the level of sequence identity isat least 90%.

In some embodiments, the γ-Netrin-binding peptide, in particular theNetrin1-binding peptide, according to the invention is fused to aheterologous peptide or polypeptide. A “heterologous peptide orpolypeptide” in the context of the invention is any peptide with alength of at least 4 amino acids, which originates from another speciesas compared to the γ-Netrin-binding peptide or which is an artificial,i.e. non-naturally occurring peptide or polypeptide. Examples of suchheterologous (poly)peptides are protein tags, in particular epitope tagssuch as the Myc-tag and the HA-tag, affinity tags such as FLAG,poly(His), chitin binding protein (CBP), maltose binding protein (MBP)and glutathione-S-transferase (GST), enzymes such as alkalinephosphatase, luciferase, horseradish peroxidase and β-galactosidase, orfluorescent proteins such as GFP, RFP and the like. Other examples aresequences that direct peptides attached thereto to specific locationsinside cells or to the extracellular space (signal sequences); specificexamples of such sequences are represented by SEQ ID NOs.: 70 and 71.

Further preferred examples of heterologous (poly)peptides areimmunoglobulins (Ig) or functional fragments of immunoglobulins, such asFv, scFv, Fab, Fab′, F(ab′)2, Fc, diabodies, minibodies, domainantibodies (dAb), camelid antibodies, nanobodies (VHH), disulfidestabilized Fv fragments (dsFv) and CDR-comprising peptides. Theimmunoglobulins or fragments thereof may be of any isotype, e.g. of theIgA-, IgD-, IgE, IgG or IgM-type. A functional structure analogous tothe isotype G of immunoglobulins (IgG) is preferred. Among these,immunoglobulins or fragments thereof of the IgG1-, IgG2-, IgG3-, orIgG4-type are preferred.

In particularly preferred embodiments, the heterologous peptide orpolypeptide is an Ig Fc fragment, for example an Fc fragment from mouse,rat, chicken, rabbit or human, with human Ig Fc fragments beingpreferred. Still more preferably, the human Ig Fc fragment is a humanIgG Fc fragment, e.g. a human IgG1, IgG2, IgG3, or IgG4 Fc fragment.

The heterologous (poly)peptide may be conjugated to the γ-Netrin-bindingpeptide directly or via a spacer of suitable length. Suitable spacersare e.g. heterologous peptide linkers having a length of from 10 to 50,preferably from 10 to 30 amino acid residues. It is further preferredfor the peptide linkers to be flexible linkers without a secondarystructure. For example, suitable peptide linkers consist of at least 80%or at least 90%, preferably at least 95% or completely of glycine and/orserine residues. Particularly suitable are peptide linkers which containa plurality of sequences SGGGG (SEQ ID NO 82).

In some preferred embodiments of the invention, the inventiveγ-Netrin-binding peptides, particularly the Netrin1-binding peptides,are competitive with Netrin receptors and can even release a γ-Netrin,e.g. human Netrin1 or zebrafish Netrin1a or 1 b, bound to a Netrinreceptor.

In a further aspect, the invention relates to a γ-Netrin-binding peptideas defined herein for use in medicine. A “use in medicine” in thecontext of the invention may be a use in therapy and/or a use indiagnostics. Preferably, the γ-Netrin-binding peptide, in particular theNetrin1-binding peptide, according to the invention is for use in humanmedicine, but it may also be used for veterinary purposes.

In particular, a γ-Netrin-binding peptide comprising (i) the sequenceEVMPTLDMALFDWTDYEDLKP (SEQ ID NO.:1), or (ii) the sequenceDVAPTFNMALFDWTDYEDMRP (SEQ ID NO.:2), or (iii) the sequenceEVMPTLDMTLFDWTDYEDMKP (SEQ ID NO.:3), or (iv) a variant thereof having asequence identity of at least 70%, at least 80%, at least 85%, at least90%, or at least 95% to SEQ ID NO.:1, 2 and/or 3, which is optionallyfused to a heterologous peptide or polypeptide, is for use in theprevention or treatment of a condition associated with, accompanied byor mediated by pathologic, particularly increased, γ-Netrin expressionor activity. Preferably, the γ-Netrin is Netrin1.

A “pathologic expression or activity” of γ-Netrin, in particularNetrin1, as referred to herein is generally meant to encompass allsituations, in which the ratio of expression and/or activity betweenDraxin and γ-Netrin and/or between γ-Netrin and at least one Netrinreceptor is abnormal.

In particular, a “pathologic expression” means any level of expression,either on the DNA or the protein level, that differs from that of anormal healthy subject. Preferably, a pathologic γ-Netrin expression maybe or may lead to a decreased γ-Netrin protein level or, morepreferably, an increased γ-Netrin protein level, particularly anincreased Netrin1 protein level, in a given (extracellular) environmentof an organism. In accordance with preferred embodiments of theinvention, the presence of an increased γ-Netrin level, particularlyprotein level, more particularly Netrin1 protein level, is defined aslying between 1 and 2 standard deviations above the average of healthyadults. Similarly, the presence of a decreased γ-Netrin protein level isdefined as lying between 1 and 2 standard deviations below the averageof healthy adults. For example, the γ-Netrin level in patient samplesmay be increased or decreased by a factor of at least 1,3 as compared tocontrol samples from healthy adults.

A “pathologic activity” as referred to herein means in particular anyactivity of a protein that is abnormal. Preferably, a pathologicγ-Netrin activity may be a decreased or increased activity, e.g. bindingto Netrin receptors with a lower or higher affinity. In accordance withpreferred embodiments of the invention, the presence of an increasedγ-Netrin activity, particularly Netrin1 activity, is defined as lyingbetween 1 and 2 standard deviations above the average of healthy adults.Similarly, the presence of a decreased γ-Netrin activity is defined aslying between 1 and 2 standard deviations below the average of healthyadults. For example, the γ-Netrin activity in patient samples may beincreased or decreased by a factor of at least 1,3 as compared tocontrol samples from healthy adults.

A control group of “healthy adults” according to the invention can bedetermined by the skilled person with routine experimentation. Forexample, “healthy adults” for use as a possible control group herein maybe healthy persons between 25 and 65 years of age, having a body massindex of 20-25, which may be sex-matched, if applicable.

The γ-Netrin-binding peptide for use as defined above may e.g. be afull-length Draxin from any vertebrate species, in particular humanDraxin, rat Draxin, mouse Draxin, zebrafish Draxin A or zebrafish DraxinB. It may also be a variant of a full-length Draxin protein,characterized in that the variant differs in at least one, at leastfive, at least ten, at least 20 or even more amino acids from thecomplete sequence of the respective full-length Draxin protein, providedit retains the γ-Netrin-, in particular the Netrin1-binding activity. Inpreferred embodiments, the peptide is however significantly shorter ascompared to full-length Draxin. For example, the peptide may have alength of up to 328, up to 324, up to 320, up to 300, up to 250, up to200, up to 150, up to 100, up to 75 or up to 50 amino acids. In somepreferred embodiments, the peptide has a length of up to 50 amino acids,e.g. 21 amino acids or 22 amino acids and is derived from zebrafishDraxinA. The peptide may or may not be fused to a heterologous peptideor polypeptide. Suitable peptides or polypeptides for fusion with theγ-Netrin-binding peptide are those described herein above, preferablyprotein tags, immunoglobulins (Ig) or functional fragments ofimmunoglobulins, wherein Ig Fc fragments, particularly human Ig Fcfragments are preferred. Still more preferably, the human Ig Fc fragmentis a human IgG Fc fragment, e.g. a human IgG1, IgG2, IgG3, or IgG4 Fcfragment.

In some embodiments of the invention, the condition associated with,accompanied by or mediated by pathologic, particularly increased,γ-Netrin, preferably Netrin1 expression or activity is ahyperproliferative disease. A hyperproliferative disease according tothe invention may be a tumor disease, a premalignant, non-neoplastic ornon-malignant hyperproliferative disorder. In particular, the conditionis a tumor disease.

An exemplary hyperproliferative disease which may be prevented ortreated using the γ-Netrin-binding peptides, particularlyNetrin1-binding peptides according to the invention is neoplasticlesions found in human inflammatory bowel disease (IBD).

Exemplary tumor diseases, which may be prevented or treated using theγ-Netrin-binding peptides, particularly Netrin1-binding peptidesaccording to the invention, are selected from the group consisting ofbreast cancer, renal cancer, liver cancer, prostate cancer, colorectalcancer, lung cancer, neuroblastoma, meningioma of the brain, pituitaryadenoma, glioma, glioblastoma, acute myeloid leukemia, sarcoma,melanoma, ovarian adenocarcinoma, renal adenocarcinoma, uterusadenocarcinoma, stomac adenocarcinoma, kidney adenocarcinoma and rectaladenocarcinoma, pancreatic cancer, inflammation driven cancers,particularly colorectal cancer, colorectal cancer associated with IBD,colorectal cancer associated with ulcerative colitis, colorectal cancerassociated with Crohn's disease, and tumors derived from inflammatorybowel disease, including metastatic and particularly aggressive forms ofthese tumor diseases.

According to one embodiment, the tumor disease is neuroblastoma.

Preferably, the tumor disease is selected from the group consisting ofpancreatic cancer, colorectal cancer, breast cancer, particularlymetastatic breast cancer, and lung cancer, particularly non-small celllung cancer.

In other embodiments of the invention, the condition associated with,accompanied by or mediated by pathologic γ-Netrin, particularly Netrin1expression or activity is a cardiovascular disease, in particularatherosclerosis.

In still other embodiments of the invention, the condition associatedwith, accompanied by or mediated by pathologic γ-Netrin, particularlyNetrin1 expression or activity is a neurological disorder, e.g. spinalcord injury.

The γ-Netrin-binding peptide, particularly Netrin1-binding peptide foruse according to the invention may, according to a further aspect of theinvention, also be present as an active agent in a pharmaceuticalcomposition together with at least one pharmaceutically acceptablecarrier. The present invention thus relates to a pharmaceuticalcomposition comprising a γ-Netrin, particularly Netrin1-binding peptideor a salt or solvate thereof and at least one pharmaceuticallyacceptable carrier. Optionally, the pharmaceutical composition furthercomprises pharmaceutically acceptable excipients and/or adjuvants.Concentrations of these carriers, excipients and/or adjuvants, if used,are in a range that is physiologically acceptable.

A “carrier” as used herein must be physiologically acceptable and retainthe therapeutic properties of the substance with which it isadministered. Standard acceptable pharmaceutical carriers and theirformulations are known to the skilled person. The carriers used willdiffer according to the administration route. Examples are magnesiumcarbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin,starch, gelatine, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting-point wax, cocoa butter, water,alcohols, polyols, glycerol, vegetable oils, buffers etc.

As exemplary excipients, disintegrators, binders, fillers, andlubricants may be mentioned. Examples of disintegrators includeagar-agar, algins, calcium carbonate, cellulose, colloid silicondioxide, gums, magnesium aluminium silicate, methylcellulose, andstarch. Examples of binders include microcrystalline cellulose,hydroxymethyl cellulose, hydroxypropylcellulose, andpolyvinylpyrrolidone. Examples of fillers include calcium carbonate,calcium phosphate, tribasic calcium sulfate, calciumcarboxymethylcellulose, cellulose, dextrin, dextrose, fructose,lactitol, lactose, magnesium carbonate, magnesium oxide, maltitol,maltodextrins, maltose, sorbitol, starch, sucrose, sugar, and xylitol.Examples of lubricants include agar, ethyl oleate, ethyl laureate,glycerin, glyceryl palmitostearate, hydrogenated vegetable oil,magnesium oxide, stearates, mannitol, poloxamer, glycols, sodiumbenzoate, sodium lauryl sulfate, sodium stearyl, sorbitol, and talc.

Usual stabilizers, preservatives, wetting and emulsifying agents,consistency-improving agents, flavour-improving agents, salts forvarying the osmotic pressure, buffer substances, solubilizers, diluents,emollients, colorants and masking agents and antioxidants come intoconsideration as pharmaceutical adjuvants.

In yet a further aspect of the invention, the γ-Netrin-binding peptide,particularly Netrin1-binding peptide for use according to the inventionmay be widely combined with other therapeutically active agents,especially to achieve a synergistic effect in therapy and/or to be ableto reduce the dosage of one or more active ingredients. Thus, thepeptide is, in some embodiments, for use together with at least oneadditional therapeutically active agent, wherein the additionaltherapeutically active agent is particularly selected from the groupconsisting of decoy Netrin receptors, cytostatic agents, cytotoxicagents, statins, antihyperlipidemic agents, anti-coagulant agents,kinase inhibitors and angiogenesis modulators. Especially preferredtherapeutic agents for combination with the γ-Netrin-binding peptide arecytostatic agents and cytotoxic agents.

A combination of a γ-Netrin-binding peptide, particularly aNetrin1-binding peptide, with at least one additional therapeuticallyactive agent can be applied either by separate administration of theactive ingredients to the patient or in the form of combination productsin which a plurality of active ingredients are present in onepharmaceutical preparation. When the active ingredients are administeredby separate administration of the active ingredients, this can be donesimultaneously or consecutively.

The peptides according to the invention, either in an isolated form oras a pharmaceutical composition will typically be administered to asubject in need thereof, in particular a human subject. They willtypically also be administered in a therapeutically effective amount,i.e. in an amount sufficient to achieve the desired effect. For example,one desired effect to be achieved by administration of γ-Netrin-bindingpeptides or anti-γ-Netrin antibodies or fragments or derivativesdescribed herein may be to block, inhibit and/or neutralize one or morebiological function of γ-Netrins, in particular of Netrin1, such as thebinding to one or more Netrin receptor(s).

Administration of suitable compositions may be effected in differentways, e.g., by intravenous, intraperitoneal, subcutaneous,intramuscular, topical, oral, intradermal, intranasal or intrabronchialadministration. Administration may also be conducted directly at thetarget site.

The necessary amount to be effective depends on a number of factors,such as the choice of the specific compound, the intended use, theadministration route and the clinical condition of the patient. Anappropriate “effective” amount in any individual case may be determinedby a skilled person in the art, e.g. a skilled physician, using routineexperimentation. An exemplary “therapeutically effective amount” of apeptide according to the invention may be about 0.01 mg to 50 mg/dose,preferably 0.1 mg to 10 mg/dose.

In yet a further aspect, the invention relates to an antibody orfragment or derivative thereof directed against Draxin, which inhibitsbinding of Draxin to γ-Netrins, in particular to Netrin1.

In yet a further aspect, the invention relates to an antibody orfragment or derivative thereof directed against Netrin1, which inhibitsbinding of at least one γ-Netrin, in particular of Netrin1, to at leastone Netrin receptor, and which is directed against an epitope betweenamino acids 285-451 of human Netrin1. In particular, the antibodyinhibits binding of at least one γ-Netrin, preferably Netrin1 to atleast one of DCC (Deleted in colorectal cancer), DSCAM, DSCAM-L1, PTPRF(protein tyrosine phosphatase, receptor-type F), NEO (Neogenin), ADORA2B(adenosine A2B), Nope (Neighbor Of Punc Eli), and members of the UNC5Hfamily (Uncoordinated-5 homologues; UNC5H1, UNC5H2, UNC5H3, UNC5H4).

The term “antibody” as used herein particularly refers to moleculescomprising at least one immunoglobulin heavy chain and at least oneimmunoglobulin light chain. Each heavy and light chain may comprise avariable and a constant domain. The antigen-binding site may be formedfrom the variable domains of a heavy and a light chain. A variableregion (also referred to as variable domain) comprises complementaritydetermining regions (CDRs), e.g. a CDR1, a CDR2 and a CDR3 region, andframework regions (FRs) flanking the CDRs. The term “complementaritydetermining region” is readily understood by the skilled person (see,for example, Harlow and Lane (eds.), Antibodies: A Laboratory Manual,CSHL Press, Cold Spring Harbor, N.Y., 1988; incorporated herein byreference in its entirety) and refers to the stretches of amino acidswithin the variable domain of an antibody that primarily make contactwith the antigen and determine antibody specificity. This region is alsoknown as the hypervariable region.

The term “(functional) antibody fragment or derivative thereof” as usedherein encompasses fragments of antibodies, especially of human orhumanized antibodies, such as portions of the above-mentioned antibodieswhich comprise at least one antigen-binding site. Examples of antibodyfragments according to the invention include Fab fragments, Fab′fragments, F(ab′)₂ fragments, Fv fragments, diabodies or single chainantibody molecules and other fragments as long as they exhibit thedesired capability of binding to their target antigen, e.g. Draxin or atleast one γ-Netrin. As exemplary single chain antibody molecules, scFvand nanobodies (VHH) are mentioned.

In case of antibodies or fragments or derivatives thereof directedagainst Draxin, only those antibodies, fragments and derivatives whichinhibit the binding of Draxin to at least one γ-Netrin, preferably toNetrin1 are encompassed by the invention.

Likewise, in case of antibodies or fragments or derivatives thereofdirected against γ-Netrin(s), only those antibodies, fragments andderivatives which inhibit the binding of at least one γ-Netrin,preferably Netrin1, to at least one Netrin receptor are encompassed bythe invention.

Such inhibition can be determined by the skilled person via routineexperimentation, e.g. via binding assays.

The term “bind” or “binding” of e.g. an antibody as used herein means anat least temporary interaction or association with or to e.g. a targetantigen, e.g. Draxin or a γ-Netrin, comprising fragments thereofcontaining an epitope.

In preferred embodiments, the antibody directed against Draxin (i.e. theanti-Draxin antibody) according to the invention binds to an epitope onDraxin, which is located between amino acid residues 209-284, preferablybetween amino acid residues 226-257, of a zebrafish Draxin, preferablyzebrafish DraxinA. More preferably, the antibody binds to an epitopebetween amino acids 232-252 of zebrafish DraxinA.

In further preferred embodiments, the anti-Draxin antibody according tothe invention binds to an epitope on Draxin, which is located in aregion of human Draxin corresponding to amino acid residues 209-284,preferably between amino acid residues 226-257, of zebrafish Draxin,preferably zebrafish DraxinA. More preferably, the antibody binds to anepitope between amino acids 222-243 of human Draxin.

In preferred embodiments, the antibody directed against γ-Netrin (i.e.the anti-γ-Netrin antibody) according to the invention binds to anepitope on Netrin1, which is located between amino acid residues 285-451(i.e. EGF1-3), preferably between amino acid residues 341-451 (i.e.EGF2-3), of human Netrin1 or a corresponding epitope in another γ-Netrinand/or another species, preferably to an epitope located in the EGF1-3domains, preferably the EGF2-3 domains of zebrafish Netrin1a and/orNetrin1b. More preferably, the antibody binds to an epitope betweenamino acids 404-451 (i.e. EGF3) of a human γ-Netrin, preferably humanNetrin1 or to an epitope located in the EGF3 domain of a zebrafishγ-Netrin, preferably zebrafish Netrin1a or Netrin1 b.

The antibody or fragment or derivative thereof according to theinvention may be derived from any antibody-producing animal species.Preferably, it is a mouse, rat or human antibody or functional antibodyfragment or antibody derivative. The antibody may be a polyclonalantibody, a monoclonal antibody, a chimeric antibody, and/or arecombinant antibody. Monoclonal antibodies, in particular human orhumanized monoclonal antibodies are preferred.

A monoclonal antibody (also referred to as mAB) is a single molecularspecies of antibody and is usually produced by creating hybridantibody-forming cells from a fusion of nonsecreting myeloma cells withimmune spleen cells. Polyclonal antibodies, by contrast, are produced byinjecting an animal (such as a rodent, rabbit or goat) with an antigen,and extracting serum from the animal. A chimeric antibody is an antibodyin which the variable domain of e.g. a murine antibody is combined withthe constant region of a human antibody. Recombinant antibodies areobtained via genetic engineering without having to inject animals. Humanantibodies according to the invention may be prepared using transgenicmice or by phage display; these methods are well known in the art.

In yet a further aspect, the antibodies or fragments or derivativesthereof according to the invention are for use in medicine (as definedabove), particularly human medicine. For example, they may be used indiagnostics to determine qualitatively or quantitatively theirrespective antigens; they may also be used as a diagnostic agent fordiseases with pathologic, in particular increased target expression.

In certain embodiments of yet a further aspect, the anti-γ-Netrinantibody (including its functional fragments and derivatives as definedherein) is for use in the prevention or treatment of a conditionassociated with, accompanied by or mediated by pathologic, particularlyincreased, γ-Netrin, particularly Netrin1 expression or activity, asdefined herein above. This condition is preferably a hyperproliferativedisease, in particular a tumor disease.

Exemplary tumor diseases, which may be prevented or treated using theanti-γ-Netrin antibody, preferably anti-Netrin1-antibody, or fragmentsor derivatives thereof according to the invention, are selected from thegroup consisting of breast cancer, renal cancer, liver cancer, prostatecancer, colorectal cancer, lung cancer, neuroblastoma, meningioma of thebrain, pituitary adenoma, glioma, glioblastoma, acute myeloid leukemia,sarcoma, melanoma, ovarian adenocarcinoma, renal adenocarcinoma, uterusadenocarcinoma, stomac adenocarcinoma, kidney adenocarcinoma and rectaladenocarcinoma, pancreatic cancer, inflammation driven cancers,particularly colorectal cancer, colorectal cancer associated with IBD,colorectal cancer associated with ulcerative colitis, colorectal cancerassociated with Crohn's disease, and tumors derived from inflammatorybowel disease, including metastatic and particularly aggressive forms ofthese tumor diseases.

Preferably, the tumor disease is selected from the group consisting ofpancreatic cancer, colorectal cancer, breast cancer, particularlymetastatic breast cancer, and lung cancer, particularly non-small celllung cancer. In another embodiment, the tumor disease is neuroblastoma.

The anti-γ-Netrin antibody, preferably anti-Netrin1-antibody or fragmentor derivative thereof may also be present in the form of apharmaceutical composition as defined above, and may also be used incombination with further pharmaceutically active agents, particularlyone or more agent selected from the group consisting of decoy Netrinreceptors, cytostatic agents, cytotoxic agents, statins,antihyperlipidemic agents, anti-coagulant agents, kinase inhibitors andangiogenesis modulators. Especially preferred therapeutic agents forcombination with the anti-γ-Netrin-antibody are cytostatic agents andcytotoxic agents.

In certain embodiments of yet a further aspect, the anti-Draxin antibody(including its functional fragments and derivatives as defined herein)is for use in the prevention or treatment of a condition associatedwith, accompanied by or mediated by pathologic, particularly decreased,γ-Netrin, particularly Netrin1 expression or activity, as defined hereinabove. Likewise, the anti-Draxin antibody (including its functionalfragments and derivatives as defined herein) may be used in theprevention or treatment of a condition associated with, accompanied byor mediated by pathologic, particularly increased, Draxin expression oractivity.

This condition may be a cardiovascular disorder, in particular acardiovascular disorder which can be prevented or treated by increasingγ-Netrin, in particular Netrin1 expression and/or activity. Exemplarycardiovascular disorders are selected from ischemia/reperfusion (I/R)injury, e.g. renal I/R injury, I/R injury of cardiac tissue; myocardialinfarction, particularly infarcts resulting from I/R injury;mitochondrial damage; neointimal formation and restenosis; vascularinjury or vascular dysfunction; vascular smooth muscle cell migrationand proliferation; apoptosis of endothelial progenitor cells, procureinduced restenosis; and hypertension.

A “pathologic expression or activity” of Draxin is generally meant toencompass all situations, in which the ratio of expression and/oractivity between Draxin and any γ-Netrin is abnormal. Particularly, apathologic Draxin expression may be or may lead to a decreased Draxinprotein level or, preferably, an increased Draxin protein level in agiven (extracellular) environment of an organism. A pathologic Draxinactivity may be an decreased or increased activity, e.g. binding to aγ-Netrin, particularly Netrin1 with a lower or higher affinity.

In yet a further aspect, the present invention relates to peptides whichbind to Draxin, in particular to human Draxin and/or zebrafish DraxinAand/or zebrafish DraxinB, with a high specificity and a high affinity.

Accordingly, the invention provides a Draxin-binding peptide comprising(i) at least 20 consecutive amino acids from the sequenceKACDCHPVGAAGKTCNQTTGQCPCKDGVTGITCNRCANGYQQSRSPIAPC IKIPIAPP (SEQ ID NO.:51) or (ii) a variant thereof having a sequence identity of at least70%, at least 80%, at least 85%, at least 90%, or at least 95% to SEQ IDNO.:51, wherein said peptide has a length of up to 580 amino acids andis optionally fused to a heterologous peptide or polypeptide.

In particular, the Draxin-binding peptide comprises at least 20, atleast 30, at least 40, at least 50 consecutive amino acids from SEQ IDNO.:51 or the complete SEQ ID NO.: 51 or a variant thereof having asequence identity of at least 70%, at least 80%, at least 85%, at least90%, or at least 95% to SEQ ID NO.:51.

In some embodiments, the peptide has a length of up to 580 amino acids,up to 500 amino acids, up to 400 amino acids, up to 300 amino acids, upto 250 amino acids, up to 200 amino acids, up to 100 amino acids, up to75 amino acids, or up to 60 amino acids. Fragments comprising up to 60amino acids are preferred according to some embodiments.

In some embodiments, the peptide with a length of up to 580 amino acidscomprises SEQ ID NO.: 51 or a variant thereof having a sequence identityof at least 70%, at least 80%, at least 85%, at least 90%, or at least95% to SEQ ID NO.: 51 and is free of any further heterologous peptides.

In other embodiments, the peptide with a length of up to 580 amino acidscomprises SEQ ID NO.: 51 or a variant thereof having a sequence identityof at least 70%, at least 80%, at least 85%, at least 90%, or at least95% to SEQ ID NO.: 51 and is fused to a heterologous peptide orpolypeptide as defined above, preferably protein tags, immunoglobulins(Ig) or functional fragments of immunoglobulins, wherein Ig Fcfragments, particularly human Ig Fc fragments are preferred. Still morepreferably, the human Ig Fc fragment is a human IgG Fc fragment, e.g. ahuman IgG1, IgG2, IgG3, or IgG4 Fc fragment.

In some embodiments, the peptide may also be fused to a heterologoussignal sequence, e.g. a human Netrin1-fragment comprising SEQ ID NO.: 51may be fused to a zebrafish signal sequence (comprising e.g. SEQ ID NO.:70 or 71), as shown in SEQ ID NOs.: 68 and 69.

In a further aspect, the invention relates to a Draxin-binding peptideas defined herein for use in medicine, particularly human medicine.

In certain embodiments of yet a further aspect, the Draxin-bindingpeptide described herein is for use in the prevention or treatment of acondition associated with, accompanied by or mediated by pathologic,particularly decreased, γ-Netrin, in particular Netrin1 expression oractivity, as defined herein above. Likewise the Draxin-binding peptidemay be used in the prevention or treatment of a condition associatedwith, accompanied by or mediated by pathologic, particularly increased,Draxin expression or activity.

This condition may be a cardiovascular disorder, in particular acardiovascular disorder which can be prevented or treated by increasingγ-Netrin, in particular Netrin1 expression and/or activity. Exemplarycardiovascular disorders are selected from ischemia/reperfusion (1/R)injury, e.g. renal I/R injury, I/R injury of cardiac tissue; myocardialinfarction, particularly infarcts resulting from I/R injury;mitochondrial damage; neointimal formation and restenosis; vascularinjury or vascular dysfunction; vascular smooth muscle cell migrationand proliferation; apoptosis of endothelial progenitor cells, procureinduced restenosis; and hypertension.

The Draxin-binding peptide for use according to the invention may,according to a further aspect of the invention, also be present as anactive agent in a pharmaceutical composition together with at least onepharmaceutically acceptable carrier. The present invention thus relatesto a pharmaceutical composition comprising an Draxin-binding peptidethereof and at least one pharmaceutically acceptable carrier and,optionally, pharmaceutically acceptable excipients and/or adjuvants.

Still a further aspect of the present invention is a method for thetreatment of a condition associated with, accompanied by or mediated bypathologic, particularly increased or decreased γ-Netrin, in particularNetrin1 expression or activity, comprising administering aγ-Netrin-binding peptide, a Draxin-binding peptide, an anti-γ-Netrinantibody and/or an anti-Draxin antibody as described herein above to asubject, particularly a human subject in need thereof. In particular,this subject suffers from a hyperproliferative disorder as definedabove.

The invention also refers to a nucleic acid molecule, preferably anisolated nucleic acid molecule, encoding a γ-Netrin-binding peptide,particularly the Netrin1-binding peptide, the Draxin-binding peptide,and the antibodies or fragments or derivatives thereof directed againstγ-Netrin, in particular Netrin1, and/or Draxin as described above. Theterm “nucleic acid molecule” encompasses a natural DNA or RNA or arecombinantly or synthetically produced DNA, RNA or LNA or arecombinantly produced chimeric nucleic acid molecule comprising any ofthese nucleic acids either alone or in combination. For example, thenucleic acid may be cDNA or genomic DNA corresponding to an entire geneor a substantial portion thereof or to fragments and derivativesthereof. The nucleotide sequence may correspond to the naturallyoccurring nucleotide sequence or may contain single or multiplenucleotide substitutions, deletions or additions. The nucleic acid mayalso be fused to another nucleic acid. The nucleic acid molecule of theinvention may be in operative linkage to an expression control sequence,i.e. to a sequence which is necessary to effect the expression of codingnucleic acid sequences. Such expression control sequences may includepromoters, enhancers, ribosomal binding sites and/or transcriptiontermination sequences. Specific examples of suitable expression controlsequences are known in the art.

The nucleic acid molecule of the invention may be located on a vectorwhich may additionally contain a replication origin and/or a selectionmarker gene. Examples of vectors are plasmids, cosmids, phages, virusesetc.

Further, the invention refers to a recombinant cell, which comprises thenucleic acid molecule as described above. The nucleic acid molecule maybe introduced into the recombinant cell by transformation, transfectionor transduction according to any method known in the art. Therecombinant cell may e.g. be a prokaryotic or eukaryotic cell.Preferably, the cell is a mammalian cell, e.g. a hamster, rabbit, orhuman cell. Preferably, the cell is a human cell.

The γ-Netrin-binding peptide, the Draxin-binding peptide, and theantibodies or fragments or derivatives thereof directed against γ-Netrinand/or Draxin of the invention may be prepared by a method, wherein thecell as described above is cultured under conditions which allowexpression of the antibody encoding nucleic acid molecule. The antibodymay be collected from the cultured cell or the culture supernatant.Preferably, the antibody is prepared from a mammalian, particularly froma human cell.

Also encompassed by the invention are salts and solvates, preferablypharmaceutically acceptable salts and solvates of the disclosedpeptides, in particular of the γ-Netrin-, preferably Netrin1-bindingpeptides and the Draxin-binding peptides.

Pharmaceutically acceptable salts may include, but are not limited to,acid addition salts and basic salts. Examples of acid addition saltsinclude chloride, sulfate, hydrogen sulfate, (hydrogen) phosphate,acetate, citrate, tosylate or mesylate salts. Examples of basic saltsinclude salts with inorganic cations, e.g. alkaline or alkaline earthmetal salts such as sodium, potassium, magnesium or calcium salts andsalts with organic cations such as amine salts.

A “solvate” is a complex of a peptide of the invention or a salt thereofwith solvent molecules, e.g. organic solvent molecules and/or water.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Draxin directly binds Netrin1.

A) AVEXIS screen results showing that the DraxinA prey protein, inaddition to the positive control bait Matn4 (+), only binds to Netrin1ain a protein library consisting of 171 bait proteins. B) Interactionnetwork of the zebrafish Netrin1 and Draxin paralogs. Consecutive screenresults show that the interaction is also conserved for the paralogs.

FIG. 2. The binding of Draxin to Netrin1 is conserved for the humanhomologs and detectable across human and zebrafish proteins.

Heatmap showing results from the AVEXIS assay. The screen has beenperformed in both bait-prey orientations using pentameric prey proteins.Absorbance at 486 nm (A486 nm) has been measured after 1-hour incubation(black: A486 nm>0.1=binding, grey: A486 nm 0.08-0.1=weak binding, 2repeats). B) Network view of the results.

FIG. 3. Draxin protein alignment using the Clustal W method.

The alignment of human, mouse, chick, and zebrafish DraxinA and DraxinBprotein shows that the N-terminal half of the protein is poorlyconserved. In the most C-terminal part of the protein a conserved10-cysteine containing region can be found, which resembles thecysteine-rich region present in Dickkopf proteins.

FIG. 4. A conserved 21aa DraxinA derived peptide is sufficient for thebinding to Netrin1a.

A) By using a set of truncated and deletion containing monomeric DraxinApreys the binding interface of DraxinA to Netrin1a has been mapped downto a 21 amino acid region (aa232-aa252). The protein fragments bindingto Netrin1a are indicated in black, weak binding ones in grey, and nonbinding fragments are depicted in white. B) and C): Using a similarapproach the DraxinA binding interface in Netrin1a has been narroweddown to the third EGF-domain containing region (aa401-aa458).

FIG. 5. Multiple species alignment of the Draxin derived 21 as peptidesequence.

The figure shows that the Netrin1 binding peptide is highly conserved invertebrates.

FIG. 6. DraxinA inhibits the binding of Netrin1a to Netrin receptors.

A) Schematic representation of the AVEXIS based competition assay. B)Purified full-length Draxin inhibits the binding of of the Netrin1a baitto the DCC-prey proteins. C) This effect can also be seen for the Unc5band Neo1 (D) netrin receptors. Equal amounts of BSA (D) are not able toinhibit the binding between Netrin and Neo1. E) Draxin is not able tointerfere with the binding of RGMc to Neo1. RGMc is another known Neo1ligand. (% binding: binding with inhibitor/binding withoutinhibitor×100%, error bars indicate mean±s.d.; n=4)

FIG. 7. A 21 amino acid fragment of Draxin is sufficient to outcompeteNetrin/Netrin receptor interactions.

A) Full-length DraxinA-hFc and the 21aa peptide fused to the hFc regionare able to outcompete DCC for binding to Netrin1a. In contrast the hFcfusion of full-length DraxinA with a deletion of aa231-252 is not ableto compete for binding. None of the 3 DraxinA-hFc fusion proteinversions is able to block the binding between other known testedreceptor-ligand pairs: (B) Cntn1a/Ptprz1b, (C) Vasn/Islr2, and (0)EphB4a/EphrinB2a. (% binding: binding with inhibitor/binding withoutinhibitor×100%, error bars show mean±s.d.; n=3)

FIG. 8. The binding of the 21aa DraxinA derived peptide is highlyspecific.

A) A pentameric DraxinA aa232-252-prey protein has been screened againsta library consisting of 141 bait proteins. We only observed binding tothe Netrin1a-bait. The positive control (+) corresponds to Matn4. B) TheDraxinA aa232-252-bait has been screened against 191 pentameric preyproteins and only showed binding to the Netrin1a prey.

FIG. 9. Draxin outcompetes receptor bound Netrin1a.

A) Scheme of the experimental design. B) The inhibitory effect ofDraxinA is not decreased by already preformed Netrin1a/DCC complexes.

FIG. 10: Protein alignment of the third EGF domain of Netrins.

Protein alignment of the third EGF domain of human Netrin1, Netrin3,Netrin4 and zebrafish Netrin1a and Netrin1b shows that this domain ishighly conserved for the γ-chain netrins but not for Netrin4 whichbelongs to the laminin1β-chain derived netrins. In agreement with theprotein alignment data our AVEXIS binding data indicate that onlyγ-chain derived Netrins can bind to Draxin.

FIG. 11: In vivo detection of the Draxin-Netrin1a interaction inzebrafish embryos.

A) Schematic illustration of the assay design. mRNAs encoding theindicated fluorophore tagged genes were injected into one-cell stagezebrafish embryos and imaged at sphere stage (4 hours post fertilization(hpf)). The imaging plane corresponded to a region approximately 15 μmbeneath the enveloping layer of the embryos.

B) Single section confocal images of the embryos. (Ba, Ba′, Ba″) embryosinjected with 100 pg Draxin-super folder GFP (sfGFP) mRNA displayeduniform distribution of Draxin-sfGFP protein in the extracellular space.In contrast injection of 100 pg of Netrin1a-sfGFP mRNA (Bb, Bb′, Bb″)resulted in dense membrane associated speckles positive forNetrin1a-sfGFP protein. In (Ba) and (Bb) memRFP has been used to labelthe cell surface. Upon co-injections of 200 pg Draxin-sfGFP mRNA and 200pg of Netrin1a-mCherry mRNA (Bc, Bc′, Bc″) Draxin-sfGFP andNetrin1a-mCherry proteins co-localize into membrane associated spots.(n=7; arrowheads point to examples of co-localization; scale barscorrespond to 10 μm).

FIG. 12: In situ detection of the Draxin-Netrin1a interaction inzebrafish using an affinity probe.

A) Schematic illustration of the experiment. ADraxin_(aa209-284)-hFc-fusion protein was generated in HEK293-6E cellsas a probe to detect endogenous netrins. Mildly fixed 48 hpf embryoswere incubated with the affinity probe (Draxin_(aa209-284)-hFc) and thesignal detection has been carried out by using a fluorophore tagged antihuman IgG antibody.

B) Results of the in situ detection experiments. The signal fromDraxin_(aa209-284)-hFc-fusion probe was detectable in the floor plateregion in wild type (also abbreviated as wt) fish (Ba), and was notdetectable in netrin1a and netrin1b double-knockdown embryos (Bb). Theshh:GFP transgenic line has been used for the experiments to visualizefloor plate cells. Arrowheads point to the Draxin_(aa209-284)-hFcderived signal. Scale bar in (Ba″), 20 μm; applies to all panels. (n>10)

FIG. 13: Heatmap depicting binding results between human DRAXIN andNetrin signaling system members.

AVEXIS was used to test pairwise binding events in both bait/preyorientations between human DRAXIN, the derived 21 amino acid peptide,human Netrin family members, and two representative Netrin receptors(DCC, UNC5B). The Matn4-bait served as an internal prey-protein controland was used for normalization of the A486 nm values; conditioned medium(CM) serves as negative control; n=3.

FIG. 14: Surface Plasmon Resonance Analysis of Draxin Binding.

Binding of Draxin to immobilised recombinant human Netrin1, UNC5B, andDCC was monitored using Surface Plasmon Resonance experiments on aBiacore 3000 instrument. See text for details.

The invention will be further illustrated by the following examples.

EXAMPLES Methods

AVEXIS Based Library Screen

To detect extracellular protein-protein interactions in ahigh-throughput manner we used the AVEXIS assay as described in Bushellet al. (Bushell et al., 2008) with minor modifications.

In brief: A zebrafish protein library enriched for mainly in neuronaltissues expressed secreted proteins and extracellular domains of cellsurface proteins has been assembled. The library consists of prey andbait proteins. Preys are composed of the extracellular domain (ECD) ofinterest followed by a CD4 tag (rat Cd4d3+4) and a pentamerizationdomain derived from the rat Comp protein followed by β-lactamase. Forthe bait proteins the ECDs are fused to a CD4 tag and a biotinylationpeptide. All proteins for the screen have been expressed by transienttransfection of Human Embryonic Kidney (HEK293-6E) cells (Durocher etal., 2002) grown in Freestyle medium (Invitrogen) containing 1% FCS.Supernatants have been harvested 6 days post transfection. The baitproteins have been dialyzed against HBS (140 mM NaCl, 5 mM KCl, 2 mMCaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4) to remove free biotin. Theproteins in the supernatants have been quantified and normalized.

For the AVEXIS screen the supernatant dilution factors have beenadjusted to values allowing faithful detection of the interactionbetween Vasn (Slit-like2) and Islr2 (Söllner and Wright, 2009) withdetermined KD of 12 μM and a very short half-life (t½ 0.16 s) (inpreparation). The bait proteins have been immobilized on streptavidincoated 96 well microtiter plates (one bait/well) and incubated for 1hour at room temperature. After 3 wash steps using HBS as wash bufferthe baits have been probed by 50 μl of normalized prey proteins. Afterone-hour incubation the non-bound preys have been washed away by 2washes with HBST (0.05% Tween) followed by two HBS washes. Then 50 μl ofnitrocefin/well (0.1 mg/ml) has been added and incubated for 1 h at roomtemperature. Then the absorbance at 486 nm of each well has beenmeasured using a μQuant spectrophotometer (BIO-TEK Instruments, INC), Asa positive control for the prey proteins we used the Matn4 ECD asmono-biotinylated bait protein. Matn4 has been shown to bind to thecoiled-coil pentamerization domain of Comp (Mann et al., 2004), which ispresent in all recombinant pentameric prey proteins of our AVEXISlibrary.

In the primary screen interactions were ‘called’ if the absorbance (at486 nm) of a well after 1 h of incubation was ≥0.1 and 3 Sigma above thestandard deviations of the mean of all wells. All interactions detectedin the primary screen have been retested in a validation screen by usingindependently produced batches of proteins.

Domain Mapping Experiments

For the binding interface mapping experiments we used monomeric preyproteins. The concentration of the monomeric preys has been determinedas described for the AVEXIS assay (β-lactamase enzymatic activity).After normalization, the monomeric preys have been screened against aset of proteins from the library to identify and remove promiscuousbinders caused for example through improper domain boundary design. Bothprey and bait orientations were tested in the domain mapping screen andtwo repeats for each orientation had been carried out.

Protein Purification

His-tagged full-length zebrafish Draxin protein (Draxin-CD4d3+4-6×His)has been expressed in HEK293-6E cells and affinitiy purified from tissueculture supernatants using HisTrap HP columns (GE healthcare). Thecorrect size of the purified protein has been checked on a protein gel.

AVEXIS Based Competition Assay

The procedure of the competition assay is based on the AVEXIS assay.Netrin1 receptors were used as prey proteins together with 6×His-taggedpurified Draxin (potential antagonist), and probed against Netrin1a baitproteins. The indicated concentrations of the potential inhibitors havebeen added together with prey proteins. For the competition tests withpurified Draxin the concentration of the Netrin receptor prey proteinshas been adjusted to an identical threshold binding concentration.

hFc Fusion Protein Normalization for the Competition Assay

We determined the concentration of ECD-hFc fusion proteins in tissueculture supernatants by ELISA, using the human IgG Fc fragment(Calbiochem) as a reference. Dilution series of the ECD-hFc containingsupernatants have been incubated over night at 4° C. on 96 well Maxisorpplates (Nunc). After 3 PBS washes the plates were blocked with 0.5% BSAcontaining PBS (1 h). After additional 2 PBS washes and 1-hourincubation with an anti-human IgG (Fc specific) antibody fused toalkaline phosphatase (SIGMA) the plates have been washed 3 times withPBS. The detection has been carried out by addition of 50 μl/well of theAP substrate p-nitrophenyiphosphate (SouthernBiotech). The substrateturnover has been determined by measuring the absorbance at 405 nm.

In Vivo Binding Assay

To reveal whether the interaction between Draxin and Netrin1a isdetectable in vivo, an mRNA overexpression assay was designed tovisualize the localization of the two proteins in zebrafish embryos.Constructs of full-length zebrafish Draxin and Netrin1a fused withfluorescent proteins were generated by using the Gateway® cloning system(Life Technologies).

Before used in the in vivo detection assay, the coding sequences of thegenerated fusion proteins were cloned into AVEXIS plasmids, expressed aspreys and tested for activity against bait proteins of the correspondingbinding partner. Following constructs were selected for the in vivobinding test: Draxin and Netrin1a C-terminally fused to superfolder-GFP(Draxin-sfGFP, Ntn1a-sfGFP) and Netrin1a C-terminally fused with mCherry(Ntn1a-mCherry). The corresponding capped mRNAs were synthesized usingthe mMESSAGE mMACHINE SP6 or T7 Transcription Kit (Ambion) according tomanufacturer instructions. For the injections, zebrafish embryos weredechorionated using 1 mg/ml Pronase (Roche, 11459643001) and theninjected with 1 nl mRNA into the cell center at one cell stage.Draxin-sfGFP or Netrin1a-sfGFP mRNAs were injected at 100 pg/embryo incombination with 10-15 pg/embryo of membrane-tagged RFP (mRFP) to labelthe cell membranes. The same amount of mRNA (100-200 pg/embryo) wasinjected in the Draxin-sgGFP and Netrin1a-mCherry coexpressionexperiments. The injected embryos have been cultured at 28.5° C. inagarose-coated dishes. At sphere stage (4 hpf), embryos were immobilizedin 1% low-melting-point agarose in glass-bottom Petri dishes with theanimal pole facing the coverslip. The imaging plane corresponded to aregion approximately 15 μm beneath the enveloping layer of the embryos.Single plane confocal images of the embryos were taken using a Zeiss LSM780 NLO microscope.

In Situ Detection of Draxin Binding Partners

A netrin binding fragment of Draxin (aa209-284) fused to the Fc regionof human IgG (Draxin_(aa209-284)-hFc) has been expressed in HEK293-6Ecells and used as an affinity probe to detect binding partners inzebrafish embryos. Draxin_(aa209-284)-hFc in situ staining has been donein whole mount wild type and netrin-1 knockdown zebrafish embryos. At 46hpf wild type zebrafish embryos were dechorionated by for 2 hours at RTincubation in 0.1 mg/ml Pronase. At 48 hpf embryos were prefixed for 10min at RT in 4% (w/v) paraformaldehyde (PFA) containing 1% Triton X-100(v/v). After 3 washes (each 20 min) with PBS+1% Triton X-100 the embryoswere blocked for 4 h at RT in PBS containing 0.2% BSA and 0.5% TritonX-100. Overnight incubation at 4° C. with HEK293-6E supernatantcontaining the Draxin-hFc fusion protein was followed by 3 short washes(10 min each) with PBST. Subsequently the embryos were post-fixed in 4%PFA (4 h at RT or overnight at 4° C.), rinsed shortly 3 times with PBSTand incubated for 4 h at RT with an Alexa Fluor 568 goat anti human IgGantibody (Invitrogen, 1:250 dilution). After 3 washes (30 min each) inPBST the embryos were mounted in glycerol for visualization using aZeiss LSM 510 microscope. A shorter version of Draxin containing theNetrin1a binding site had to be used because the full-length version ofDraxin-hFc caused uniform background staining in zebrafish embryosprobably by unspecific binding to glycosaminoglycans (GAGs) present oncell surfaces. A series of additional control ECD-hFc proteins have beentested, only Draxin_(a209-284)-hFc displayed binding to theextracellular space of the floor plate.

Knockdown of netrin1a and netrin1b in Zebrafish Embryos

Morpholino antisense oligonucleotides (Gene-Tools) have been used togenerate zebrafish with reduced netrin protein expression levels. Thefollowing morpholino sequences have been used to knockdown ntn1a(5′-ATGATGGACTTACCGACACATTCGT-3′, SEQ ID NO.: 80) and ntn1b(5′-CGCACGTTACCAAAATCCTTATCAT-3′, SEQ ID NO.: 81). In previous studiesboth morpholinos had been shown to efficiently knockdown thecorresponding genes (Kastenhuber et al., 2009; Suli et al.; 2006).

Surface Plasmon Resonance (SPR)

Surface Plasmon Resonance (SPR) experiments were performed on a Biacore3000 (GE Healthcare) at 25° C. using a SA sensor chip in 0.01 M HEPES,pH 7.4, 0.15 M NaCl, 0.005% Surfactant P20 (HBS-P) running buffer at aflow rate of 30 μl/min. The instrument was used according tomanufacturer's instructions.

Example 1: DraxinA Physically Interacts with Netrin1a

Using a protein-protein interaction screen assay, designed to identifydirect interactions within a protein library consisting of secretedproteins and extracellular domains of cell surface proteins (Bushell etal., 2008), we carried out a large-scale screen involving more than40,000 binding experiments. The library we used for the screen wasstrongly enriched for zebrafish proteins known to be expressed in thedeveloping nervous system. During this screen we identified a novelinteraction between two secreted proteins with known function in axonguidance, Netrin1a and DraxinA. In the primary screen a DraxinA preyprotein has been tested for binding against a library consisting of 171bait proteins, including a positive control. The DraxinA prey proteinspecifically bound to the Netrin1a bait (FIG. 1A) and did not bind toany additional proteins of the library. The interaction has beenconfirmed in both bait-prey orientations in a validation screen usingnew protein samples. Interestingly, both netrin1 and draxin areduplicated in zebrafish. In subsequent binding assays we were able toshow that the Netrin1/Draxin interaction is also conserved for theparalogs Netrin1b and DraxinB (FIG. 1B).

The AVEXIS assay is able to detect very transient and weak interactionsdue to the avidity effect caused by the use of pentameric prey proteins.Hence, in order to test whether the interaction between Netrin1a andDraxin is transient or rather stable we used monomeric prey proteins andprobed them against the corresponding binding partners. Using thisapproach, we confirmed the interaction between Netrin1a and Draxinsuggesting that this interaction is based on strong binding between thetwo proteins.

Example 2: The Interaction Between Draxin and Netrin1 is Conserved forthe Human Homologs

Next we asked the question whether the interaction between Netrin1 andDraxin is conserved. By using the corresponding human homologs NTN1 andDRAXIN we were able to show that the interaction is indeed conserved. Inaddition, we observed that zebrafish Netrin1a was able to bind to humanDraxin and vice versa (FIG. 2). This strongly indicates that this newlyidentified interaction is conserved within vertebrate speciesunderscoring the biological relevance of this interaction.

Example 3: The Netrin1 Binding Region of Draxin has been Mapped Down toa 21aa Motif

Next, we narrowed down the region in DraxinA required for binding toNetrin1a. Zebrafish DraxinA consists of 360 amino acids (aa). The first23 as are part of the signal peptide. This sequence is followed by apoorly conserved N-terminal half of the protein (FIG. 3). In contrast,the C-terminal half of the protein is highly conserved and ends with a10 cysteine-containing domain (aa285-aa360). In terms of cyteine spacingthis domain is similar to domains present in the Wnt antagonist Dkk1(Glinka et al., 1998).

To map down the Netrin1a binding region in DraxinA we generated a seriesof DraxinA truncations and deletions and tested them for binding againstNetrin1a (FIG. 4). Using this approach we were able to narrow down thebinding region to a 21aa DraxinA protein fragment. In addition, afull-length version of DraxinA lacking these 21aa completely lost theability to bind to Netrin1a. Additional removal of 5aa from theN-terminal or C-terminal end of the 21aa stretch caused a dramaticreduction of the binding ability to Netrin1a. Interestingly, theNetrin-binding 21aa stretch (aa232-252) of DraxinA is highly conservedcross vertebrate species (FIG. 5). It is noteworthy that, this conserved21aa region is also highly specific for Draxin and cannot be found inother proteins.

Example 4: Netrin1a Domain Mapping

Netrin1a is a multi-domain containing protein composed of 603 aminoacids. It consists of a laminin N-terminal domain (LamNT) encoded byamino acid 44-282 followed by 3 laminin-type epidermal growthfactor-like domains (aa284-450), and a C-terminal domain (C345C) encodedby amino acid 486 to 594. In order to map the DraxinA binding region inNetrin1a we generated a set of truncated Netrin1a fragments and probedthem in the AVEXIS assay for binding against DraxinA. Using thisapproach we were able to narrow down the binding region to a fragmentconsisting of amino acid 401-458 (FIG. 4B). This fragment encodes thethird laminin-type EGF domain. The third EGF-domain of Netrin1a ishighly conserved in vertebrate Netrin1 homologs. For example, only asingle amino acid exchange is present in this domain between zebrafishNetrin1a (CDCHPVGAAGKTCNQTTGQCPCKDGV TGITCNRCANGYQQSRSPIAPC; SEQ ID NO.:64) and human Netrin1 (CDCHPVGAAGKTCNQTTGQCPCKDGVTGITCNRCAKGYQQSRSPIAPC;SEQ ID NO.: 65) proteins.

Interestingly, the third EGF domain of Netrin1 has recently been shownto be required for Netrin receptor binding (Finci et al., 2014; Xu etal., 2014). These findings offer a mechanistic explanation for ourobserved competition assay results.

Example 5: DraxinA is Able to Inhibit the Binding of Netrin1a to NetrinReceptors

By using an AVEXIS-based competition assay (FIG. 6 A) we tested whetherthe binding of DraxinA to Netrin1a has an influence on Netrins abilityto bind to Netrin receptors. First we confirmed that we reliablydetected the binding of Netrin1a to Netrin receptors of the DCC/Neo1 andUnc5 families with the AVEXIS method. We also tested whether Draxin isable to bind to the corresponding Netrin receptors. Contrary to previousfindings (Ahmed et al., 2011), we were not able to detect direct bindingbetween Draxin and any of the tested Netrin receptors using the AVEXISplatform. This has also been confirmed by a recent publication (Haddicket al. 2014). In the competition assay the extracellular domains (ECDs)of Netrin receptors have been used as prey proteins together withpurified full length DraxinA and probed for binding against Netrin1abait proteins. Using this strategy, we observed a DraxinA concentrationdependent inhibition of the binding between Netrin1a and Netrinreceptors (FIG. 6 B,C,D). The inhibition is specific for DraxinA.Furthermore, DraxinA (FIG. 6 E) is not able to block the binding of RGMcto Neo1, another reported ligand of the corresponding receptor (Bell etal., 2013).

Example 6: The 21Aa DraxinA Fragment Fused to the Human Fc Region of IgGis Sufficient to Block the Binding of Netrin1a to Netrin Receptors

Next we assayed whether the 21aa DraxinA fragment fused to the human Fctag (Draxin_(aa232-252)-hFc) is sufficient to outcompete Netrin/Netrinreceptor interactions.

We compared the effect of Draxin_(aa232-252)-hFc with full-lengthDraxin-hFc and a version of DraxinA where the 21aa Netrin1 binding motifhas been deleted (DraxinAΔ_(aa231-252)-hFc) in the competition assay fortheir ability to interfere with the binding of Netrin1a to Dcc. Theresults show that Draxin_(aa232-252)-hFc has a similar efficiency ininhibiting the binding of Netrin1a to Dcc as the DraxinA full-lengthversion (Draxin-hFc). The DraxinA version with the 21aa deletion(DraxinAΔ_(aa231-252)-hFc) is not able to compete for binding toNetrin1a (FIG. 7). In addition we used our set of different Draxin-hFcproteins to test whether they have an effect on other known interactions(Cntn1a/Ptprz1b, Vasn/Islr2, EphB4a/EphrinB2a). None of the 3 Draxin-hFcversions was able to inhibit any of the tested interactions (FIG. 7B,C,D).

Taken together, our results show that the 21aa region is necessary forthe competition and that the Draxin_(aa232-252)-hFc fusion protein isalso sufficient to outcompete Netrin receptors for Netrin1a binding.

Example 7: The Binding of the 21Aa DraxinA Fragment to Netrin1a isHighly Specific

In order to determine the binding specificity of the 21aa DraxinApeptide we used the AVEXIS assay and screened the 21aa fragment as baitand prey against proteins from our library. In this screen against a setof 141 bait proteins the DraxinA_(aa232-252)-prey only bound to theNtn1a-bait and to the positive control bait Matn4 (FIG. 8A). Similarresults we obtained for the DraxinA_(aa232-252) bait protein, which onlyinteracted with the Ntn1a-prey in a screen against a set of 191different prey proteins (FIG. 8B). These findings indicate that thebinding of this short 21aa peptide to Netrin1a is highly specific.

Example 8: DraxinA Outcompetes Receptor Bound Netrin 1a

Netrin1 binds with high affinity (K_(d)'s in the low nM range) to itsreceptors of the DCC- and Unc5-family (Leonardo et al., 1997). Hence, weasked the question whether already bound Netrin1a could be displacedfrom the receptors by DraxinA. In order to do so we carried out anAVEXIS based competition assay and tested three different settings (FIG.9A). In one experimental setting Netrin1a-baits were preincubated withpurified DraxinA before addition of the DCC prey. In the second set ofexperiments the Netrin1a-baits were incubated with a mixture of DraxinAand DCC prey proteins, and in the third set of experimentsNetrin1a-baits have been preincubated with DCC preys followed by theaddition of purified DraxinA as inhibitor. We did not observe adifference in the %-binding response between these 3 sets ofexperiments. These findings show that already formed DCC/Netrin1acomplexes can be disrupted by the addition of DraxinA. These findingsindicate that DraxinA has a higher affinity for Netrin1a than the Netrinreceptor DCC.

Example 9: In Vivo Detection of the Draxin-Netrin Interaction inZebrafish Embryos

To independently confirm the Draxin/Netrin1a interaction and to testwhether both proteins are able to interact in vivo, we made use oftransient protein overexpression experiments in zebrafish embryos. mRNAsencoding Draxin fused to superfolder GFP (Draxin-sfGFP) and Netrin1atagged with mCherry (Netrin1a-mCherry) or superfolder GFP(Netrin1a-sfGFP) have been injected into one-cell stage zebrafishembryos. The distribution of the corresponding fluorophore taggedproteins has been analyzed in sphere stage zebrafish embryos (4 hourspost fertilization) (FIG. 11A). At this developmental stage theextracellular space width between the cells is very large, ideallysuited to visualize the localization of secreted proteins. Uponinjection of mRNA encoding Draxin-sfGFP we observed an evenlydistributed signal outside the cells in the extracellular milieu of 4hpf zebrafish embryos (FIG. 11Ba). In contrast thereto, the distributionof Netrin1a-sfGFP was restricted to cell surface sub-domains (FIG.11Bb).

When Draxin-sfGFP was coexpressed with Netrin1a-mCherry, Draxin-sfGFPre-located to Netrin1a-mCherry positive membrane associated densities(FIG. 11Bc). This indicated that localized Netrin1a-mCherry was able tocapture diffusible Draxin-sfGFP.

The mRNA overexpression experiments showed that Draxin and Netrin1a areable to interact with each other in vivo. To further support this weused another strategy aiming to detect the distribution of endogenousDraxin interaction partners at developmental stages relevant for axonguidance decisions. From our binding assay with monomeric prey proteinswe already had hints that the interaction between Draxin and Netrin isof high-affinity. Thus, we fused a netrin-binding fragment of theDraxin-ECD (aa209-284) to the human Fc region to generate an affinityprobe. First, we tested this probe on zebrafish embryos from differentdevelopmental stages. After very gentle fixation the embryos wereincubated with HEK293-6E cell supernatants containing the recombinantsoluble Draxin_(aa209-284)-hFc protein.

Using an Alexa-Fluor 568 anti human IgG antibody to detect in situ boundDraxin_(aa209-284)-hFc we only detect a signal in close proximity to thefloor plate (FIG. 12A, 12Ba). Because floor plate cells express Netrin1aand Netrin1b, we had indications that the signal detected by using theDraxin affinity probe indeed corresponds to in the extracellular spacelocalized netrin. To prove this observation, we compared 48 hpf wtembryos with netrin1a and netrin1b double-knockdown embryos (FIG. 12Ba,12Bb). In double-knockdown embryos the signal from the bound affinityprobe was barely detectable compared to non-injected siblings,indicating that the Draxin_(aa209-284)-hFc probe indeed detected netrin.Taken together, the results from our mRNA overexpression and Draxinaffinity probe experiments provide strong evidence that Draxin and Ntn1aare able to interact in vivo in zebrafish embryos.

Example 10: Human DRAXIN/Netrin-Signaling Network

To determine the binding specificity of DRAXIN/Netrin interactions, wecarried out a pairwise binding screen between human DRAXIN and humanNetrin family members. Except Netrin-5, we included all human Netrinfamily members consisting of two secreted γ-Netrins (Netrin-1 andNetrin-3) and one secreted (Netrin-4) and two GPI-linked β-Netrins(Netrin-G1 and Netrin-G2) in our binding study. Human DRAXIN and a 21amino acid Netrin binding fragment derived thereof (SEQ ID NO.: 1) boundto Netrin-1 and Netrin-3 but not to human β-Netrin family members (FIG.13).

These experiments confirm for human proteins the Draxin/γ-Netrin bindingspecificity within the Netrin family and showed that the human 21 aminoacid DRAXIN fragment (SEQ ID NO.: 1), like its zebrafish counterpart(SEQ ID NOs.: 3), is sufficient for binding.

Example 11: Validation of the Draxin/Netrin1 Interaction by SurfacePlasmon Resonance

Recombinant human Draxin was purchased from R&D systems. Biotinylatedrecombinant human UNC5B, Netrin1 and DCC were produced recombinantlyusing the described mammalian expression system (HEK293-6E).Biotinylated proteins were immobilised on the SA coated sensor chip andDraxin was injected sequentially in increasing concentrations (0 nM, 1.2nM. 2.3 nM, 4.7 nM, 9.4 nM, 18.8 nM) for 3 min. Dissociation was allowedfor 5 min in HBS-P. Binding was monitored and an interaction of Draxinto immobilised Netrin-1 was observed with a binding constant KD ofapproximately 20 to 100 nM. No binding of Draxin to UNC5B and DCC wasdetected (FIG. 14).

REFERENCES

-   Ahmed G, Shinmyo Y, Ohta K, Islam S M, Hossain M, Naser I B, Riyadh    M A, Su Y, Zhang S, Tessier-Lavigne M, Tanaka H: Draxin inhibits    axonal outgrowth through the netrin receptor DCC. J Neurosci. 2011    Sep. 28; 31(39):14018-23.-   Arakawa H: Netrin-1 and its receptors in tumorigenesis. Nat Rev    Cancer. 2004 December; 4(12):978-87.-   Bell C H, Healey E, van Erp S, Bishop B, Tang C, Gilbert R J,    Aricescu A R, Pasterkamp R J, Siebold C: Structure of the repulsive    guidance molecule (RGM)-neogenin signaling hub. Science. 2013 Jul.    5; 341(6141):77-80.-   Bushell K M, Söllner C, Schuster-Boeckler B, Bateman A, Wright G J:    Large-scale screening for novel low-affinity extracellular protein    interactions. Genome Res. 2008 April; 18(4):622-30.-   Castets M, Broutier L, Molin Y, Brevet M, Chazot G, Gadot N, Paquet    A, Mazelin L, Jarrosson-Wuilleme L, Scoazec J Y, Bernet A, Mehlen P:    DCC constrains tumour progression via its dependence receptor    activity. Nature. 2012; 482(7386):534-7.-   Delloye-Bourgeois C, Brambilla E, Coissieux M M, Guenebeaud C,    Pedeux R, Firlej V, Cabon F, Brambilla C, Mehlen P, Bernet A:    Interference with netrin-1 and tumor cell death in non-small cell    lung cancer. J Natl Cancer Inst. 2009 Feb. 18; 101(4):237-47.-   Durocher Y, Ferret S, Kamen A: High-level and high-throughput    recombinant protein production by transient transfection of    suspension-growing human 293-EBNA1 cells. Nucleic Acids Res. 2002    Jan. 15; 30(2):E9.-   Finci L I, Kruger N, Sun X, Zhang J, Chegkazi M, Wu Y, Schenk G,    Mertens H D, Svergun D I, Zhang Y, et al.: The Crystal Structure of    Netrin-1 in Complex with DCC Reveals the Bifunctionality of Netrin-1    As a Guidance Cue. Neuron 2014; 83(4):839-849.-   Fitamant J, Guenebeaud C, Coissieux M M, Guix C, Treilleux I,    Scoazec J Y, Bachelot T, Bernet A, Mehlen P: Netrin-1 expression    confers a selective advantage for tumor cell survival in metastatic    breast cancer. Proc Natl Acad Sci USA. 2008 Mar. 25; 105(12):4850-5.-   Glinka A, Wu W, Delius H, Monaghan A P, Blumenstock C, Niehrs C:    Dickkopf-1 is a member of a new family of secreted proteins and    functions in head induction. Nature. 1998 Jan. 22; 391(6665):357-62.-   Islam S M, Shinmyo Y, Okafuji T, Su Y, Naser I B, Ahmed G, Zhang S,    Chen S, Ohta K, Kiyonari H, Abe T, Tanaka S, Nishinakamura R,    Terashima T, Kitamura T, Tanaka H: Draxin, a repulsive guidance    protein for spinal cord and forebrain commissures. Science. 2009    Jan. 16; 323(5912):388-93.-   Haddick P C, Tom I, Luis E, Quiñones G, Wranik B J, Ramani S R,    Stephan J P, Tessier-Lavigne M, Gonzalez L C: Defining the Ligand    Specificity of the Deleted in Colorectal Cancer (DCC) Receptor. PLoS    One. 2014 Jan. 6; 9(1):e84823.-   Kastenhuber E, Kern U, Bonkowsky J L, Chien C B, Driever W, and    Schweitzer J: Netrin-DCC, Robo-Slit, and heparan sulfate    proteoglycans coordinate lateral positioning of longitudinal    dopaminergic diencephalospinal axons. J Neurosci 2009; 29:8914-8926.-   Kwan K M, Fujimoto E, Grabher C, Mangum B D, Hardy M E, Campbell D    S, Parant J M, Yost H J, Kanki J P, and Chien C B: The Tol2kit: a    multisite gateway-based construction kit for Tol2 transposon    transgenesis constructs. Dev Dyn 2007; 236:3088-3099.-   Leonardo E D, Hinck L, Masu M, Keino-Masu K, Ackerman S L,    Tessier-Lavigne M: Vertebrate homologues of C. elegans UNC-5 are    candidate netrin receptors. Nature. 1997 Apr. 24; 386(6627):833-8,-   Mann H H, Ozbek S, Engel J, Paulsson M, Wagener R: Interactions    between the cartilage oligomeric matrix protein and matrilins.    Implications for matrix assembly and the pathogenesis of    chondrodysplasias. J Biol Chem. 2004 Jun. 11; 279(24):25294-8.-   Mehlen P, Delloye-Bourgeois C, Chédotal A: Novel roles for Slits and    netrins: axon guidance cues as anticancer targets? Nat Rev Cancer.    2011 March; 11(3):188-97.-   Moore S W, Tessier-Lavigne M, Kennedy T E: Netrins and their    receptors. Adv Exp Med Biol. 2007; 621:17-31.-   Paradisi A, Creveaux M, Gibert B, Devailly G, Redoulez E, Neves D,    Cleyssac E, Treilleux I, Klein C, Niederfellner G, Cassier P A,    Bernet A, Mehlen P: Combining chemotherapeutic agents and netrin-1    interference potentiates cancer cell death. EMBO Mol Med. 2013    December; 5(12):1821-34.-   Paradisi A, Maisse C, Coissieux M M, Gadot N, Lépinasse F,    Delloye-Bourgeois C, Delcros J G, Svrcek M, Neufert C, Fléjou J F,    Scoazec J Y, Mehlen P: Netrin-1 up-regulation in inflammatory bowel    diseases is required for colorectal cancer progression. Proc Natl    Acad Sci USA. 2009 Oct. 6; 106(40):17146-51-   Söllner C, Wright G J: A cell surface interaction network of neural    leucine-rich repeat receptors. Genome Biol. 2009; 10(9):R99.-   Suli A, Mortimer N, Shepherd I, and Chien C B: Netrin/DCC signaling    controls contralateral dendrites of octavolateralis efferent    neurons. J Neurosci 2006; 26:13328-13337.-   Xu K, Wu Z, Renier N, Antipenko A, Tzvetkova-Robev D, Xu Y,    Minchenko M, Nardi-Dei V, Rajashankar K R, Himanen J, et al.: Neural    migration. Structures of netrin-1 bound to two receptors provide    insight into its axon guidance mechanism. Science 2014; 344:    1275-1279.

The invention claimed is:
 1. γ-Netrin-binding peptide comprising (i) thesequence EVMPTLDMALFDWTDYEDLKP (SEQ ID NO.: 1), (ii) the sequenceDVAPTFNMALFDWTDYEDMRP (SEQ ID NO.: 2), (iii) the sequenceEVMPTLDMTLFDWTDYEDMKP (SEQ ID NO.: 3), or (iv) a variant thereof havinga sequence identity of at least 70%, at least 80%, at least 85%, atleast 90%, or at least 95% to SEQ ID NO.:1, 2 and/or 3, wherein saidpeptide has a length of up to 150 amino acids and is fused to aheterologous peptide or polypeptide.
 2. The peptide according to claim1, wherein said peptide is selected from the group consisting of SEQ IDNO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11,SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14 and SEQ ID NO.: 15 or avariant thereof having a sequence identity of at least 70%, at least80%, at least 85%, at least 90%, or at least 95% thereto.
 3. The peptideaccording to claim 1, which is fused to a functional fragment of animmunoglobulin (Ig).
 4. The peptide according to claim 1 in combinationwith a carrier suitable for use in human medicine.
 5. The peptideaccording to claim 2, wherein said peptide comprises a sequence selectedfrom the group consisting of SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.:12, SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15, or a variantthereof having a sequence identity of at least 70-95% thereto.
 6. Thepeptide according to claim 5, wherein said variant has a sequenceidentity of at least 95% thereto.
 7. The peptide according to claim 5,wherein said sequence is SEQ ID NO.:
 15. 8. A method for disruptingγ-Netrin/Netrin receptor interactions, comprising administering aγ-Netrin-binding peptide to a cell, wherein said γ-Netrin-bindingpeptide has a length of up to 150 amino acids, is fused to aheterologous peptide or polypeptide and comprises a sequence selectedfrom the group consisting of: (i) the sequence EVMPTLDMALFDWTDYEDLKP(SEQ ID NO.: 1), (ii) the sequence DVAPTFNMALFDWTDYEDMRP (SEQ ID NO.:2), (iii) the sequence EVMPTLDMTLFDWTDYEDMKP (SEQ ID NO.: 3), and (iv) avariant thereof having a sequence identity of at least 70% to SEQ IDNO.:1, 2 and/or
 3. 9. The peptide according to claim 3, wherein thefunctional fragment of an Ig is an Ig Fc fragment.
 10. The peptideaccording to claim 9, wherein the Ig Fc fragment is a human Ig Fcfragment.
 11. The peptide according to claim 10, wherein the human Ig Fcfragment is a human IgG Fc fragment.